Composition for Use in the Prevention and Treatment of Cardiovascular Diseases

ABSTRACT

The invention provides a composition the prevention and treatment of cardiovascular disease, wherein said composition compounds obtained from palm oil mill effluents, in particular from vegetative liquor from the milling of palm oil fruit.

FIELD OF INVENTION

The present invention relates to the use of a composition for theprevention and treatment of cardiovascular disease, in particular theinvention relates to a composition obtained based on extracts obtainedfrom palm oil effluent, comprising vitamin E, phenolic flavanoids andcombinations thereof, for the preparation of a means for the preventionand treatment of cardiovascular disease, particularly atherosclerosis.

BACKGROUND OF INVENTION

Atherosclerosis is an exemplary of a cardiovascular disease (CVD) thatrequires life long management in both prevention and treatment. It isrecently considered the leading cause of death in South East Asia whichaccounts for approximately 4 million fatal cases each year. Thisparticular disease is characterized by the hardening and thickening theinner lining of an artery with deposits that consist of fattysubstances, cholesterol, cellular waste products, calcium and fibrin.Consequently, if not treated, the buildup known as plaque or atheroma,which embedded the walls of arteries will eventually block the arteries,and is the main contributory factor for heart attack, chest pain orstroke.

The main carrier in relation to the occurrence of cholesterol and itsmain source of damaging accumulation and blockage as mentioned above inthe arteries is low-density lipoproteins (LDL). Generally, LDL carriescholesterol to peripheral tissues and accordingly passes through theendothelium that causes further development of plaques and thereforeforms arterial wall cholesterol. Conclusively, patients with high amountof LDL have significantly high risk of atherosclerosis.

From the above, and recognizing the fact that by reducing or loweringthe amount of LDL in patients may significantly assist to reduce theoccurrence of atherosclerosis, the development of scientificallyvalidated medicaments and treatments have been primed over the yearsbased on this vital factor.

Ongoing scientific advancements include several chemically developeddrugs that inhibit or lower the production of cholesterol; such drugsfor instance include statins; and pharmaceutical compositions.Alternative approaches at present also include the consumption of juicesor substances containing high level of antioxidants, for instance thepomegranate juice. The presence of antioxidants can assist to neutralizefree radical damage. Free radicals are highly reactive chemicalsubstances that can damage cellular materials, and therefore causesmajor degenerative illnesses including cardiovascular disease andcancer.

Additionally, botanical or plant based compositions have also played amajor role in providing effectual remedies in relation to cardiovasculardisease.

The major constituents in plant extract having beneficial propertiescomprise of antioxidants which have been widely known for treatment andprevention of a range of cholesterol related disease, wherein examplesof antioxidants in plants are phenolics acids and flavanoids. Otherplant based components or minerals with astronomical medicinal benefitswhich may be extracted from plants include potassium, calcium andmagnesium, wherein the said minerals are well recognized for preventingand managing disorders such as hypertension, cardiovascular disease, anddiabetes.

Following the above, the use of natural plant extracts havingantioxidant properties instead of synthetic antioxidants for treatmentof cardiovascular disease is now gaining momentum.

An exemplary of an excellent source of two major phytochemicals namelyvitamin E (tocopherols and tocotrienols) and carotenoids, both of whichare fat soluble, is the oil palm fruit. Palm vitamin E has been reportedto act as a potent biological antioxidant, protecting against oxidativestress and the atherosclerotic process.

It is the primary objective of the present invention to provide acomposition for preparing a medicament for the treatment of acardiovascular disease, based on plant extract.

It is further an objective of the present invention to provide acomposition for preparing a safe medicament for the treatment of acardiovascular disease, said composition containing palm oil phenolicsand palm vitamin E. The potential benefit is to inhibit the formation ofplaque and thus inhibit the development of atherosclerotic lesions.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-D are the graphs showing the plasma lipid profiles of therabbits in the four treatment groups; and

FIG. 2A-D are the graphs showing all rabbits in this experiment whichwere observed to have developed atherosclerotic lesions after feeding onthe artherogenic diet for 100 days.

SUMMARY OF INVENTION

A composition for use in the preparation of a medicament for theprevention and treatment of cardiovascular diseases, said compositioncomprising components extracted from palm oil mill effluents.

In another aspect of the present invention, there is provided acomposition for use in the prevention of cardiovascular diseases, saidcomposition further comprising Vitamin E.

Further, the present invention relates to the use of therapeuticallyeffective amount of a composition along with or without vitamin E, inthe preparation of a medicament for preventing or inhibitingcardiovascular disease in an individual by administering to anindividual in need thereof.

DETAILED DESCRIPTION

The present invention relates to the identification of excellentremedial effects for the prevention and treatment of cardiovasculardisease, particularly atherosclerosis, based on palm phenolics andvitamin E, and thus the production of a composition on the same.

A water soluble crude extract rich in flavonoids and phenolic acids wassuccessfully recovered from the vegetative liquor generated from themilling of oil palm fruits. The antioxidant activity of the palmphenolics extract in in vitro and in vivo systems has been reported byBalasundram (2006). This extract has been tested and confirmed to haveantioxidant properties and potent protective effects against cancer andatherosclerosis in animal and cell culture model systems.

The potential role of vitamin E to protect against free radical-mediateddiseases has been the subject of many studies. There is now a growinginterest in the nutritional and physiological properties of vitamin E inpalm oil especially the tocotrienols. Epidemiological studies havelinked dietary intake of vitamin E and other antioxidants with reducedrisk of coronary heart disease (Rimm et al., 1993) and ischemic stroke(Bonner et al., 1995) and with a decrease in carotid artery thickness(Kritchevsky et al., 1995). Hodis et al. (1995) reported thatantioxidant vitamins slowed the progress of coronary arteryatherosclerosis, and Theriault et al. (2002) found thatdelta-tocotrienol was the most effective vitamin E for reducing theendothelial expression of adhesion molecules and adhesion on monocytes,thus reducing the risk of developing atherosclerosis.

The following examinations show that compared to the control diet,inclusion of palm vitamin E significantly reduced the extent of fibrousplaque in these animals. This finding is consistent with the observationby Kritchevsky et al. (2000) who reported that red palm oil containingboth vitamin E and carotene, resulted in the least severeatherosclerotic lesions development compared to refined, bleached,deodorized (RBD) palm oil or randomized palm oil, and suggested that thecarotenoids and vitamin E may protect against atherosclerosis. Similareffects was also observed from another study by Wilson et al. (1978),who found that addition of 1% (w/w) Vitamin E to the diet caused amarked reduction in aortic and coronary atherosclerosis in rabbits. Theprotective effect of vitamin E on cholesterol-induced atherosclerosiswas also reported in an earlier study (Haiman, 1960). On the other hand,some researchers found that vitamin E had no effect on either plasmalipid parameters nor atherogenesis in the rabbit (Dam, 1994) whileothers reported that vitamin E improved some lipid parameters but didnot inhibit the atherosclerotic plaque formation (Ismail et al., 2000).

Accordingly, in accordance to the preferred embodiments of the presentinvention, and based on the working example which will be describedshortly, palm phenolics either on their own (AO) or in combination withvitamin E (AOE) has shown a significantly superior inhibitory effects ondevelopment of atherosclerotic lesions were observed in both the AO andAOE groups compared to the CTR and VIT E.

For the purpose of this invention, plasma antioxidant capacity wasmeasured by two different methods i.e the FRAP and ABTS⁺. Resultsobtained from the ABTS⁺ scavenging assay, showed no significantdifference in all the four groups. Results from the FRAP assay alsoappeared to be not statistically significant, though plasma antioxidantcapacity of the AO group and AOE group tended to be higher than that ofthe CTR group.

The methods and results obtained based on respective assays to determinethe effectiveness of the said compounds in palm oil vegetation will bedescribed herein.

BEST MODE FOR CARRYING OUT THE INVENTION Working Examples Materials andMethods 1. Animals, Diets and Experimental Design

Thirty-two male New Zealand White rabbits aged 4-5 months were randomlydivided into 4 different treatment groups of 8 animals each. The animalswere housed individually in stainless steel cages and maintained in atemperature controlled room (25° C.-28° C.) with a 12-hour daylightcycle. All animals were fed ad-libitum on an atherogenic diet for 100days. Additionally, the animals were also provided vitamin E, palmphenolics or a combination thereof. The four experimental groups wereclassified as follows:

-   -   1. High Fat Atherogenic Diet−Control (CTR)    -   2. High Fat Atherogenic Diet+Vitamin E (VIT E)    -   3. High Fat Atherogenic Diet+Palm Phenolics (AO).    -   4. High Fat Atherogenic Diet+Vitamin E+Palm Phenolics (AOE).

The atherogenic diets were formulated as shown in Table 1. Dietscontained 35% energy from fat.

TABLE 1 Composition of formulated rabbit atherogenic diet. Ingredientsg/kg diet Casein 250.0 Corn starch 200.0 Dextrose 193.0 Cellulose 150.0Mineral Mix 40.0 Vitamin Mix 10.0 Choline Bitartrate 2.5 DL-Methionine3.0 Cholesterol 1.5 Dietary Fat 150.0

The dietary fat used was designed to be atherogenic, and comprised:saturated fatty acids 67.1% (C12:0+C14:0=54.9%); monounsaturated fattyacids 15.7% (C18:1) and polyunsaturated fatty acids 12.0% (Table 2). Thefatty acid composition of the dietary oil was determined as their methylesters using gas chromatography (Sundram et al., 1997).

In diets of the VIT E and AOE group, palm vitamin E, rich intocotrienols was used as the vitamin E source, and 1250 mg of thisvitamin E was added to each kg of dietary fat. The final vitamin Econtent in the VIT E pellet diet was 237.0 mg/kg, whereas the basepellet diet (fed to CTR and AO groups) had 50 mg/kg vitamin E, primarilyfrom the oil used in the diet formulation. Palm phenolics were providedto the AO and AOE group as their drinking fluid containing totalphenolics at 1500 mg GAE/L. During the feeding trial, animals werecontinuously monitored for their food intake, water consumption andweight gain. The consumption of the palm phenolics of the AO and AOEgroup was measured over a 14-day continuous duration and was found to beapproximately 150 mg GAE/day/animal.

TABLE 2 Content of major fatty acids (%) in the experimental diets.Carbon No. Dietary Fat SFA 67.14 12:0 40.38 14:0 14.50 16:0 9.72 18:02.40 20:0 0.14 PUFA 12.02 18:2(n − 6) 11.88 18:3(n − 3) 0.14 MUFA 15.6818:1(n − 9) 15.68

At the end of the feeding trial, all animals were sacrificed. Prior tosacrifice the animals were fasted overnight, anesthetized with a mixtureof katemin and zoletil (0.1 mL/kg body weight) and 30 mL blood was drawnby heart puncture. Plasma was prepared by centrifugation at 3000 g for20 minutes and stored at −80° C. until analyses. The animals were thenoverdosed with sodium pentobarbital before autopsy to remove variousorgans of interest such as liver, heart, lung and kidneys. The aortasystem was carefully traced, dissected and cleaned of adherentadventitial tissue. The aorta was then cut open and preserved in 10%formalin solution before staining with oil Red-O for quantification ofatherosclerotic lesions.

2. Biochemical Analyses and Measurements of Atherogenic Indices.

All laboratory analyses listed below are current and standard in mostrelevant laboratories.

Plasma Lipid Analysis

Plasma lipids (TC, triglycerides (TG), HDL-C), were analyzed usingenzymatic assay kits (Roche Diagnostics GmbH, Mannheim, Germany) as permanufacturer's protocol on the clinical chemistry autoanalyzer,Roche/Hitachi 902.

Plasma Antioxidant Status

Plasma antioxidant status was measured by two methods; the 2,2′-azinobis(3-ethylbenzothiazoline) 6-sulfonic acid radical cation (ABTS⁺)decolorization assay and ferric reducing ability of plasma (FRAP).

The ability of rabbit plasma to scavenge the ABTS⁺ was measured usingthe method of Re et al. (1999) as adapted by Balasundram (2006).Essentially this assay measures the ability of antioxidants in theplasma to scavenge preformed ABTS⁺ produced by the oxidation of ABTS bypotassium persulfate. The intensely coloured ABTS⁺ is relatively stable,but in the presence of an antioxidant, it is readily reduced to thecolourless ABTS²⁻. The loss of absorbance at 734 nm after 6 min is takenas a measure of the ABTS⁺ scavenging activity. Standard ABTS⁺-scavengingcurves were constructed using6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox) and theABTS⁺ scavenging capacity of the plasma is reported in terms of mgTrolox equivalents/mL (mg TE/mL).

The ferric reducing antioxidant power of plasma was determined using themethod of Firuzi et al. (2005) as adapted by Balasundram (2006). Theoriginal FRAP assay was developed by Benzie & Strain (1996) as a test tomeasure the ferric reducing ability of plasma, i.e. the ability toreduce a ferric-2,4,6-tripyridyl-s-triazine (Fe³⁺-TPTZ) toferrous-2,4,6-tripyridyl-s-triazine (Fe²⁺-TPTZ) via an electron transfermechanism. This reduction resulted in the production of an intenselyblue coloured reduced complex, with maximum absorption at 593 nm (Benzie& Strain, 1996).

Histopathological Examination

The aorta was stained with oil Red-O and quantification of theatherosclerotic lesions was undertaken by using an imaging analysissoftware (IDT-Solution). Lesions were categorized as shown in Table 3below:

TABLE 3 Lesions characterization. Type of Lesions CharacteristicsFibrous Plaque Raised nodular lesions, continuous, intense red, whitehard visible to naked eyes Fatty Plaque Raised distinct lesions,intensely stained red Fatty streak Lipid accumulation, stained light redLesion Free No plaques or streaks

Statistical Analyses

AU data were analyzed using the analysis of variance (ANOVA) and thepost hoc Tukey HSD to test differences between dietarygroups/treatments. Differences were considered statistically significantat p<0.05.

Results

Mean body weights of the rabbits, prior to and at the end of the study,were not significantly different between all dietary groups after 100days feeding (Table 4). This indicates that the experimental diet didnot have any adverse outcomes on the normal growth curves of theexperimental animals.

TABLE 4 Animal body weights after 100 days treatments. CTR VIT E AO AOEInitial wt (g) 1871.43 ± 48.80  1857.14 ± 78.68  1860.97 ± 214.121822.27 ± 146.9  Final wt (g) 2229.43 ± 369.30 432.37 ± 171.39 2185.87 ±752.94 2122.47 ± 375.10 n = 8 animals/group No significant differencesbetween the treatments

At sacrifice, the weight of the liver, heart, lung and kidneys showed nosignificant difference between the treatment groups (VIT E, AO, AOE) andcontrol group (Table 5),

TABLE 5 Effect of antioxidants on various organs of rabbits at autopsyCTR VIT E AO AOE Liver 72.63 ± 11.34 61.83 ± 8.71 67.90 ± 12.63 64.46 ±11.54 Heart 4.11 ± 0.83  4.45 ± 0.60 4.30 ± 0.71 4.15 ± 0.94 Lung 7.56 ±1.63  7.20 ± 1.03 8.54 ± 1.57 7.26 ± 1.30 Kidney 9.60 ± 0.99 10.49 ±1.59 11.72 ± 1.78  9.65 ± 2.42 n = 8 animals/group No significantdifferences between the treatments

The plasma lipid profiles of the rabbits in the four treatment groupsare shown in FIG. 1A-D. These results do not show any significantdifference between groups. It must be noted that these plasma sampleswere highly lipaemic. Nanji (1984) has reported that measurement oflipid profile of lipaemic samples is problematic as the lipaemiainterferes with a variety of clinical chemistry analyses. Hence, furtherpretreatment of the samples was required to obtain more accurateresults. Results were plotted as FIG. 1, whereby the effect of thetreatments on plasma lipid profiles in rabbits can be observed. (A:Total cholesterol, B:LDL-cholesterol, C:HDL-cholesterol,D:Triglyceride). Data are presented as means±sd from 8 animals/group.

All rabbits in this experiment were observed to have developedatherosclerotic lesions after feeding on the atherogenic diet for 100days (FIG. 2A-D). The CTR group had significantly higher fibrous plaque(FIG. 2A) score compared to the other three groups (VIT E, AO and AOE).In the AOE group, all animals did not show occurrence of fibrous plaquesand this was significantly different (lower) compared to the CTR, theVIT E and AO groups. Fatty plaques (FIG. 2B) were significantly higherin both the CTR and VIT E groups compared to the AO and AOE treatments.In addition, the lowest fatty plaque score was apparent with the AOEtreatment. Fatty streaks were highest in the VIT E treated animalsfollowed by the CTR group (FIG. 2C). AO and AOE groups had significantlylower occurrence of fatty streaks compared to CTR and VIT E groups.Lesion free area (FIG. 2D), denoting the total area that was not alteredmorphologically (i.e. free of fatty streaks, fatty and fibrous plaques),was significantly higher following the AOE and AO treatments compared tothe VIT E and CTR diets. This suggests protective effects due to AO andAOE treatments compared to the CTR and VIT E treatments, againstoccurrence of atherosclerosis.

FIG. 2 shows the development of atherosclerotic lesions (A: FibrousPlaque, B: Fatty Plaque, C: Fatty Streaks, D: Lesion Free) after 100days treatments. Values are means±SD (n=8 rats in each group) Means withcommon superscripts are significantly different (p<0.05).

To measure the antioxidant capacity of the rabbits plasma, two methodswere used i.e the ABTS⁺ decolorization assay and FRAP assay. Though theFRAP results for AO (12.32±5.69 mg TE/mL) and the AOE group (10.53±4.84mg TE/mL) were comparatively higher than that of the Control group(7.18±2.66 mg TE/mL), the differences were not statistically significant(Table 6). Similarly, there were no significant differences in theplasma ABTS⁺ scavenging capacity of all the four groups. The use of twomethods for measurement of antioxidant activity was based on thesuggestion by Frankel & Meyer (2000) and Verhagen et al. (2003) on theneed to evaluate antioxidant activity by using different methods. Thesetwo methods were chosen as they are amongst those most commonly used tomeasure the antioxidant capacity due to their rapidity of analysis, easeof use and high sensitivity

(Maisuthisakul et al., 2007).

TABLE 6 Effect of the treatments on the antioxidant capacity in therabbit plasma CTR VIT E AO AOE FRAP  7.18 ± 2.66  6.17 ± 1.15 12.32 ±5.69 10.53 ± 4.84 ABTS 74.95 ± 6.75 77.14 ± 4.88 75.73 ± 4.84 76.39 ±5.12

In this study, the AO and AOE groups given palm phenolics as theirdrinking fluid did not exhibit significant changes in their plasma lipidprofile (TC, TG, LDL-C and HDL-C), though the HDL-C level was observedto be slightly higher (statistically not significant) than the CTR andVIT E groups. However, these animals (AO and AOE groups) hadsignificantly reduced incidence of atherosclerosis (i.e less fattystreaks and plaques) than the CTR and VIT E groups. Similar observationwas also reported by Wang et al. (2005) that no significant changes wereseen in plasma lipid parameters between the control and any of theexperimental groups at the end of the 12 week feeding duration. Theseauthors found that dealcoholized red wine containing known amounts ofresveratrol suppressed atherosclerosis in hypercholesterolemic rabbitsand concluded that the phythochemicals in red wine can suppressatherosclerosis without affecting plasma lipid levels. In another rabbitstudy, Chen et al. (2005) fed a high-cholesterol diet for 10 weeks torabbits, supplemented with or without mulberry fruit extract at twodifferent concentrations (i.e. either 0.5% or 1%), and showed asignificant decreased in plasma TC, LDL and TG levels.

The effect of palm phenolics in this study in inhibiting the formationof fibrous plaque was obvious in both the AO and AOE groups compared tothe CTR and VIT E group, suggesting that palm phenolics may potentiallyinhibit atherosclerosis. This finding is in agreement with the findingsof Fuhrman et al. (2005), who reported that fresh grape powderattenuated atherosclerotic development by reducing macrophages uptake ofoxidized LDL and reduced macrophages cholesterol accumulation inapolipoprotein E-deficient (E⁰) mice. These authors found no changes inthe lipid profiles of the E⁰ mice. Similarly, although no changes wereseen in plasma LDL-C or HDL-C upon feeding of red wine to E⁰ mice,reduced progression of lesions was reported by Hayek et al. (1997).Other recent animals studies also reported that consumption ofpomegranate juice by mice (Kaplan et al., 2001), grape extract byhamsters (Auger et al., 2004). Similarly, consumption red wine,dealcoholized wine and wine polyphenols was found to reduceatherosclerotic lesions in E⁰ mice, hamsters and rabbits (Manach et al.,2005). These results therefore show that the phenolics from palm couldbe similarly protective against atherosclerosis tendencies.

The compound of this invention may be prepared independently, in dosageform as described above, and can also be prepared combined together ascombination product.

The dosage form as used herein above includes any suitable vehicle forthe administration of medications known in the pharmaceutical art,including, by way of example, capsules, tablets, syrups, elixirs andsolutions for parenteral injection with specified ranges of drugconcentrations.

These examples are not intended, however, to limit or restrict the scopeof the invention in any way and should not be construed as providingconditions, parameters, reagents or starting materials which must beutilized exclusively in order to practice the present invention.

1-5. (canceled)
 6. A composition for use in the prevention and treatmentof atherogenic diseases, comprising extracts from oil palm oil milleffluents, wherein the composition exhibits inhibitory effects in theformation of fatty and fibrous plaque and atherosclerotic lesions in amammal.
 7. A composition as claimed in claim 6, wherein the compositionfurther comprises Vitamin E.
 8. A composition as claimed in claim 7,wherein the composition comprises phenolics and flavanoids extractedfrom oil palm oil mill effluents.
 9. The composition as claimed in claim6, wherein the composition further provided with pharmaceuticallyacceptable carriers
 10. Use of a therapeutically effective amount of acomposition comprising extracts from palm oil mill effluents in theprevention and treatment of atherogenic diseases in a mammal in needthereof, characterized in that the composition reduces and inhibits theformation of fatty and fibrous plaque and inhibits the development ofatherosclerotic lesions in said mammal.
 11. A method for inhibiting theformation of fatty and fibrous plaques and atherosclerotic lesions inthe treatment of atherogenic diseases in a mammal comprisingadministering a composition comprising extracts from palm oil milleffluents.